L-threonine has been produced by microorganism fermentation, and it has been prepared using mutant strains induced through screening process by treating, with a mutation inducer, wild type strains of microorganism belonging to the genus Escherichia, Corynebacterium, Brevibacterium, and Serratia. For example, Korean Patent 115393 describes a method for screening microorganisms producing L-threonine by treating a microorganism belonging to the genus Escherichia with N-methyl-N′-nitro-N-nitrosoguanidine, which is a kind of random mutation inducer, to give tolerance to purine analogs, such as 6-dimethylaminopurine, 9-azaadenine, and 8-azadiaminopurine, etc. Korean Patent 168719 describes a method for screening microorganisms producing L-threonine from wild type strain of Serratia sp. through rendering tolerance to lysine analog S-(2-aminoethyl)-L-cysteine, and rifampicin, an antibiotic using a mutation inducer. Moreover, Japanese Patent Publication 224684/83 describes a method of using a microorganism which belongs to Brevibacterium sp., has tolerance to S-(2-aminoethyl)-L-cystein and α-amino-β-hydroxyvalerate, and has a nutritional requirement for L-isoleucine and L-lysine.
Meanwhile, technologies for developing more improved L-threonine-producing microorganisms by introducing site-specific gene substitution, gene amplification and distruction, etc., into L-threonine-producing microorganisms developed by random mutation as gene recombinantion technology develops, are being reported. For example, Korean Patent 397423 describes a method for preparing L-threonine using a microorganism in which at least one copy of phosphoenolpyruvic acid carboxylase (ppc) gene and threonine operon are inserted into a specific site in a chromosomal DNA of L-threonine-producing microorganisms prepared by repetitive mutation induction and screening process to have methionine requirement, tolerance to threonine analogus (α-amino-β-hydroxyvalerate), tolerance to lysine analogus (S-(2-aminoethyl)-L-cystein), and tolerance to isoleucine analogus (e.g., α-aminobutyric acid), using genetic engineering techniques. In addition to that, many technologies for the development of L-threonine-producing microorganisms by applying gene recombination to a mutant microorganism as described above, have been also reported (US 2005/0032178, US 2004/0214294, U.S. Pat. No. 5,939,307).
However, the above-described methods have several critical disadvantages as they were developed on the basis of microorganisms prepared by treating them with a mutation inducer inducing random mutation. Treatment with a mutation inducer and microorganism screening enabled the development a strain capable of producing L-threonine in high yield, but screened microorganisms have various physiological characteristics, such as a decline in the growth rate of a strain compared to its parent strain, a decline in sugar consumption rate, and a decline in tolerance to external environmental change, which is disadvantageous for industrial production of amino acids due to many random mutations inevitably resulted from the treatment with a mutation inducer. Moreover, they have disadvantages in that there are many problems in additional strain development due to many mutants produced by treatment with a mutation inducer and there is a limitation in improving their productivity.
Accordingly, as total chromosome sequences of microorganisms were identified due to the development of genetic engineering, recently, new attempts, excluding random mutation, to overcome the above mentioned problems of random mutation have been made. A study on the development of a strain producing lysine in high yield by screening major mutants advantageous for producing lysine using comparative genomics study between lysine-producing Corynebacterium sp. developed by repetitive random mutation and a wild type strain of Corynebacterium sp., has been reported (Ohnishi, J. et al., Appl. Microbiol. Biotechnol., 58:217, 2003; Hayashi, M. et al., Appl. Microbiol. Biotechnol., 72:783, 2006).
Moreover, Veronika et al. reported that after ilvA and panB were inactivated using Corynebacterium sp., and operon (ilvBNC) involved in L-valine biosynthesis was amplified, 130 mM L-valine was produced by disrupting feedback inhibition in ilvN gene (Veronika et al., Appl. Environ. Microbiol., 71:207, 2005). Also, it was reported that 86 mM L-serine was produced by rational design using Corynebacterium sp. (Peters-Wendisch et al., Appl. Environ. Microbiol., 71:7139, 2005). All of the reports are study results on strain development obtained by carrying out only site-specific mutation by rational design excluding random mutation, but they have very poor industrial applicability except for the above mentioned lysine-producing microorganism. Moreover, it has not yet been reported that L-threonine was produced at high yield by strain development using rational design.
Therefore, there is an urgent need to develop a strain producing L-threonine in high yield by ration design methods, which can overcome the disadvantages of strain development by the existing random mutation method.
Accordingly, the present inventors have made extensive efforts to develop a mutant microorganism which can overcome disadvantages of microorganisms prepared by the existing random mutation method, and as a result, constructed a mutant microorganism producing L-threonine using only site-specific mutation, and confirmed that a high concentration of L-theronine can be produced at high yield using the mutant microorganism, thereby completing the present invention.